Use of p-aminophenol derivatives for the preparation of pharmaceutical compositions useful in the treatment of neurodegenerative diseases

ABSTRACT

A therapeutic method comprising treating a patient suffering from a neurodegenerative disease with a therapeutically effective quantity of a p-aminophenol derivative.

This application is a 371 of PCT/EP97/02708 filed May 15, 1997.

The present invention concerns the use of p-aminophenol derivatives forthe preparation of pharmaceutical compositions useful in the treatmentof neurodegenerative diseases.

More particularly, the present invention concerns the use of derivativesof p-aminophenol and prodrugs thereof for the preparation ofpharmaceutical compositions useful in the treatment of neurodegenerativediseases which contain an inflammatory component with production in locoof inflammatory markers such as proteins of the complement andcytokines.

Still more particularly, the present invention concerns the use ofcompounds having the formula ##STR1## wherein R₁ is hydrogen or loweracyl, and

R₂ is hydrogen or lower alkyl

and the use of prodrugs thereof.

Typical examples of such compounds are phenacetin (R₁ ═COCH₃ ; R₂ ═C₂H₅), also known as acetophenetidine, and paracetamol (R₁ ═COCH₃ ; R₂═H), also known as acetaminophen or N-acetyl-p-aminophenol.

Typical examples of prodrugs are propacetamol (B. Bannwarth et al., "Br.J. Clin. Pharmac.", 34, 79-81, 1992), acetaminosalol (R. Q. Brewster,"J. Am. Soc.", 40, 1136, 1918) and its glycine ester (I. M. Kovach etal., "J. Pharm. Sci.", 64, 1070, 1975). With regard to use of the term"prodrug", the reader is referred to "Burger's Medicinal Chemistry andDrug Discovery--Vol. 1; Principles and Practice--5th ed.--Ed. Manfred E.Wolff--pp. 172-178".

It is already known that these compounds possess analgesic andantipyretic actions comparable to those of aspirin, whilst unlikeacetylsalycylic acid they possess no significant anti-inflammatoryaction (Insel A. P. in: "The Pharmacologic basis of therapeutics"Goodman Gilman A. et al. ed., pp. 656-659, Pergamon Press, 1990).

Because of its general toxicity and side effects, phenacetin iscurrently little used for therapeutic purposes.

Paracetamol however is well tolerated and presents no significant sideeffects in low therapeutic doses from 0.5 to 1 g, 3 or 4 times a day.

Paracetamol is rapidly absorbed and is distributed evenly throughout themajor part of the tissues of the organism, including the brain. Theplasma concentration reached within a few minutes of oral administrationof 1 g paracetamol is approximately 10 mg/l (66.2 μM) and increases toapproximately 1.5 mg/l after 6 hours. The tissue concentration issubstantially the same as the plasma concentration (Prescott L. F., "Br.J. Clin. Pharmacol.", 10 (suppl. 2), 291-298S, 1980).

The mode of action of paracetamol is not yet fully known. It is howevergenerally agreed that its activity is dependent on its ability toinhibit prostaglandin production mainly in the central nervous system.This action would appear to explain the analgesic and antipyreticeffects of paracetamol. The absence of action on peripheralcycloxygenase could, on the other hand, be responsible for its lack ofanti-inflammatory action (Clissold, S. P., "Drugs", 32 (suppl. 4),46-59, 1986).

Alzheimer's disease is a pathology characterised by progressiveneurodegeneration leading to dementia. Autopsy examination constantlyreveals certain typical histological markers such as plates due toexcessive deposition of β-amyloid and loss of neurons and/or synapses(Selkoe D. J. et al., "Neuron", 6, 487-493, 1991).

In recent years the presence of inflammation markers in cerebral samplesfrom patients suffering from Alzheimer's disease has been documentedwith increasing frequency. Proteins typical of inflammatory processessuch as antigens of histocompatibility, certain cytokines and theirreceptors, Fc receptors, certain proteins of the acute phase andproteins of the classic pathway of activation of the complement togetherwith their receptors have in fact been identified in amyloid plates orin the region of such plates (Dickson D. W. et al., "Glia", 7, 75-83,1993). It has also been demonstrated that such inflammatory proteins aresecreted in loco by glial cells and microglial cells (Hugh Perry V. etal., "Trends Neurol. Sci.", 7, 268-273, 1993). A minor incidence of thispathology in patients suffering from rheumatoid arthritis who needanti-inflammatory drugs for long periods of time has also been provedwith adequate clarity (McGeer P. L. et al., "Neurology", 42, 447-449,1992; Breitner J. S. et al., "Neurology", 44, 227-232, 1994; Rogers J.et al., "Neurology", 43, 1609-1611, 1993).

In particular, a double blind study of 44 patients suffering fromAlzheimer's disease treated with indomethacin for 6 months has shown anotable slowing of the progression of dementia in comparison withcontrol subjects treated with a placebo (Schnabel J., "Science", 260,1719-1720, 1993). These observations indicate that the "cerebralinflammatory" phenomena typical of Alzheimer's disease are not simply areaction to the pathological phenomenon already in progress, but thatthe said cerebral inflammatory phenomena themselves become a pathogenicfactor of primary significance.

J. C. S. Breitner et al. (Neurobiology of aging, Vol. 16(4), 1995, pages523-530) refer about a retrospective investigation on subjects who hadtaken paracetamol for treating pain and fever. Although, thepharmaceutical preparations previously administered to the subjects hadnot been manufactured to treat the Alzheimer disease, this investigationhad the aim of discovering whether the treatment with paracetamol hadcause a statistically significant delay in the onset of the Alzheimerdisease. The result was negative.

WO-A-9528153 discloses a group of 11 specific benzamide compounds fortreating neurodegenerative diseases. The activity of these compounds issaid to be unexpected and not predictable based on the fact that other,structurally closely related materials lack activity. Slight structuralchanges are reported to yield large differences in efficacy andtoxicity.

It has now unexpectedly been found that the compounds of formula (I) andprodrugs thereof are useful in the treatment of neurodegenerativediseases in which an inflammatory component is present with in locoproduction of proteins of the complement and cytokines, despite the factthat such compounds possess no anti-inflammatory action.

In particular, the said compounds inhibit the production of proteins ofthe complement and cytokines by glial brain cells and allow asignificant reduction of the inflammatory phenomena typically present inthe brain of patients suffering from Alzheimer's disease.

The purpose of the present invention therefore consists of the use of acompound having the formula ##STR2## wherein R₁ is hydrogen or loweracyl, and

R₂ is hydrogen or lower alkyl,

and prodrugs thereof for the preparation of a pharmaceutical compositionuseful in the treatment of neurodegenerative diseases in which there ispresent an inflammatory component with in loco production ofinflammatory markers such as proteins of the complement and cytokines.

A typical example of such disorders is Alzheimer's disease.

In the course of the present description and of the claims which follow,the expression "proteins of the complement" indicates a complex ofapproximately 20 proteins which make up the complement system, and whichpossess an inflammatory action of cascade catalysis type. Activation ofthe system, which can take place in two ways known as the direct oralternative activation pathways, leads, in the end, to cellular lysis.

Some of the factors (proteins) are designated with the letter "C"followed by a number, and the most important and abundant component isthe factor known as C3 (I. M. Roitt, in: Immunologia; fisiologia,patologia o clinica; italian ed. by C. E. Grossi and D. Zarcone, page 7,Edi-Ermes, Milano, 1990) [Immunology; physiology, pathology or clinic;Italian edition by C. E. Grossi and D. Zarcone, page 7, Edi-Ermes,Milan, 1990]. In experiments for the purpose of assessing possibleproduction of proteins of the complement therefore, only C3 isdetermined and is considered indicative of the entire complex ofproteins of the complement.

The cytokines are a family of proteins which have the function ofenabling communication between the various cell types which form part ofthe immune system and consequently of regulating the various aspects ofthe immune response, including inflammatory phenomena. Amongst thecytokines predominately involved in inflammatory response, interleukin-6(IL-6) performs a role of primary importance: such a cytokine is in factproduced by numerous cell types (macrophages, B and T lymphocytes,endothelial cells) and controls immune response, acute phase reactionsand haematopoiesis. Raised levels of IL-6 are regarded as indicative ofan inflammatory reaction taking place and have been found in numerouspathologies which have an inflammatory component, such as rheumatoidarthritis and systemic lupus erythematosus.

The daily dose of the compound according to formula (I) can vary withina rather wide range depending on known factors such as the type andseverity of the disease, the patients body weight, the pharmaceuticalcomposition used, the method of administration, the number of dosageforms administred daily and the effectiveness of the compound used.

The optimum dose can nevertheless easily be established by an expert inthe field by routine procedures.

Generally speaking, the daily dose will be between 0.01 and 200 mg/kg.

In the particular case of paracetamol, the daily dose will preferably beof from 10 to 100 mg/kg or, even more preferably, of from 15 to 75mg/kg.

Examples of suitable methods of administration are: oral, rectal andinjection.

Depending on the administration methods chosen, the pharmaceuticalcompositions according to the present invention may be in solid form,for example tablets, coated tablets, capsules and controlled releasecompositions or semi-solid form, for example suppositories, or in liquidform for example sterile solutions. Examples of controlled releasecompositions are those which include liposomes or vesicles (capable ofreleasing the pharmacologically active compound in a controlled manner)and layered tablets comprising layers with variable disintegrationspeeds.

In addition to the usual excipients, the compositions may includeadditives suitable for pharmaceutical use such as preservatives,stabilisers, surfactants, emulsifiers, salts to regulate osmoticpressure, buffers, flavourings, sweeteners, colourings and the like.

The compositions according to the present invention may contain othercompatible active principles which may be therapeutically useful whenused simultaneously. Such compounds include: antioxidants such asacetylcysteine, vitamin E, glutathione and methionine, calciumantagonists, glutamate antagonists for NMDA receptors such asdextromethorphan, acetylcholine receptor agonists, in particularmuscarine or nicotine agonists, and pharmaceuticals capable of acting onthe nervous system by increasing acetylcholine levels at synaptic level(acetylcholinesterase inhibitors). Examples of suitable compounds whichare active on the cholinergic system are tacrine, physostigmine,neostigmine, heptastigmine, donepezil (E-2020), metriphonate andSDZ-ENA-713.

The pharmaceutical compositions according to the present invention canbe produced by conventional chemicopharmaceutical methods comprisingmixing, granulation and compression, when necessary, or various mixturesand dissolution of the ingredients depending on which is the mostsuitable to produce the required product.

The following examples serve to illustrate the present invention, butare not limitative.

EXAMPLE 1

Paracetamol and indomethacin activity on complement production in theT98G cell line

The effect of paracetamol on the production of complement factors hasbeen studied using the human glioblastoma T98G cell line. The said lineis in fact capable of constitutively producing factor 3 of thecomplement (C3) and this synthesis is further increased in the presenceof various stimuli such as interleukin-1 beta (IL-1β). Indomethacin hasbeen used as the reference drug.

The cells (2×10⁶ /ml) were incubated for 48 hours in the presence ofincreasing concentrations of paracetamol (10-100-500-1000 μM) orindomethacin (0, 1-1-10 μM) both in the presence and in the absence ofIL-1β (50 U/ml). On completion of incubation, the culture medium washarvested and the quantity of C3 secreted by the cells in the variousconditions was measured. C3 was assayed in a sandwich-typeimmunoenzymatic test, in which two anti-C3 antibodies of differentspecies were used as well as an antiserum labeled with an alkalinephosphatase specific for one of the two anti-C3 antibodies. The secretedC3 was quantified using a standard curve prepared on the basis ofpurified C3.

The results (Table 1) show that paracetamol inhibits the secretion of C3with a Cl₅₀ of approximately 1 mM both when the cells are stimulatedwith interleukin-1 beta and in the absence of the said cytokine.Indomethacin was found to be active in reducing the secretion of thecomplement in this test but, because of its toxicity in the cellularsystem used, it was not possible to increase its concentration above 10μM, at which dose it shows a 35% inhibiting activity.

These results indicate that both paracetamol and indomethacin arecapable of reducing secretion of factors of the complement which occursin the brain in pathological conditions.

These observations testify in favour of the use of paracetamol inpathologies in which significant production of factors of the complementexists in the central nervous system, such as, for example, Alzheimer'sdisease.

                  TABLE 1                                                         ______________________________________                                        Activity of Paracetamol and Indomethacin on                                     Production of Complement in the T98G Cell Line                                                 Dose     Inhibition %                                      ______________________________________                                        Cell Stimulation with IL-1β                                                Paracetamol 1000 μM 47.5                                                    500 μM 39.8                                                                100 μM 23.6                                                                10 μM 0.1                                                                 Indomethacin 10 μM 35.1                                                     1 μM 21.7                                                                  0.1 μM 26.9                                                               Cell not Stimulated with IL-1β                                           Paracetamol 1000 μM 60.8                                                    500 μM 37.4                                                             ______________________________________                                    

EXAMPLE 2

Activity of paracetamol and indomethacin on the production ofinterleukin-6 (IL-6) in the cell line T98G stimulated with β-amyloid

The effect of paracetamol on the production of IL-6 was studied usingthe human cell line glioblastoma T98G. The said line is in fact capableof constitutively producing IL-6 and this synthesis is augmented in thepresence of aggregated β-amyloid. Indomethacin was used as the referencedrug.

The cells (2×10⁶ /ml) were incubated for 48 hours in the presence ofincreasing concentrations of paracetamol (10-1000 μM) or indomethacin(0.1-10 μM) in the presence of β-amyloid (50 μM) aggregated byincubation at 37° C. for 6 days. At the end of the experiment theculture medium was harvested and the quantity of IL-6 produced in thevarious conditions was assayed using a commercial immunoenzymatic kit(Biotrak IL-6, Amersham, UK).

The results (Table 2) show that paracetamol inhibits production of IL-6with a Cl₅₀ of slightly over 1 mM. The inhibiting activity ofindomethacin on IL-6 is of from 10 to 20%.

These results indicate that paracetamol is capable of reducing thesecretion of IL-6 occurring in the brain in the presence of β-amyloid, acondition typical of Alzheimer's disease, this observation thereforejustifies the use of paracetamol in this pathology.

                  TABLE 2                                                         ______________________________________                                        Activity of Paracetamol and Indomethacin on                                     the Production of IL-6 in the T98G Cell Line                                    Cell Stimulation with β-amyloid                                                             Dose     Inhibition %                                  ______________________________________                                        Paracetamol        1000   μM 41.2                                             500 μM 20.5                                                                100 μM 6.7                                                                 10 μM 11.4                                                                Indomethacin 10 μM 11.5                                                     1 μM 17.6                                                                  0.1 μM 21.1                                                             ______________________________________                                    

EXAMPLE 3

Activity of paracetamol and indomethacin on the production ofprostaglandins in the T98G and J774.2 cell lines

The benefit of paracetamol in cerebral inflammatory pathologies has beenconfirmed by other experiments designed to demonstrate that paracetamolpossesses a selectivity of action in respect of brain cells which is notfound for other anti-inflammatory drugs, such as indomethacin.

Parallel experiments were conducted in which the ability of paracetamoland indomethacin to inhibit the production of prostaglandins produced bythe glioblastoma T98G cell line or by the J774.2 cellular macrophagiccell line was assessed.

The test with T98G cells stimulated with IL-1β was conducted asdescribed in the previous Example 1. At the end of incubation theculture medium was harvested and the quantity of E2 prostaglandinspresent was measured. The prostaglandins were assayed by means of acommercially available immunoenzymatic test (AMERSHAM).

The results given in Table 3 show that paracetamol and indomethacin areboth capable of inhibiting production of prostaglandins in the glialcells of the central nervous system in a significant manner.

                  TABLE 3                                                         ______________________________________                                        Activity of Paracetamol and Indomethacin on                                     Production of prostaglandins in the T98G cell line                              Cell Stimulation with IL-1β                                                                Dose      Inhibition %                                  ______________________________________                                        Paracetamol       1000   μM  99                                               500 μM 96.9                                                                100 μM 88.5                                                                10 μM 26.7                                                                Indomethacin 10 μM 99.1                                                     1 μM 98.4                                                                  0.1 μM 99.3                                                             ______________________________________                                    

Parallel to the above experiment, J774.2 (2×10⁶ /ml) macrophagic cellswere stimulated with lipopolysaccharide (LPS) (1 μg/ml) for 12 hours.After 3 washes with physiological solution, the cells were incubated for45 minutes together with increasing concentrations of paracetamol(0.1-100 μM) or indomethacin (0.01-1 μM). At the end of incubation thesupernatants were harvested and the prostaglandin E2 content wasmeasured using the previously described test.

The results given in Table 4 indicate that paracetamol, contrary toindomethacin, has little inhibiting activity on production ofprostaglandins in macrophages, and hence in cells situated outside thecentral nervous system.

                  TABLE 4                                                         ______________________________________                                        Activity of Paracetamol and Indomethacin on                                     Production of Prostaglandins in the J744.2 Cell Line                            Cell Stimulation with LPS                                                                       Dose      Inhibition %                                  ______________________________________                                        Paracetamol       100    μM  11.4                                             10 μM 12.5                                                                 1 μM 6.4                                                                   0.1 μM 5.3                                                                Indomethacin 1 μM 87.4                                                      0.1 μM 61.1                                                                0.01 μM 35.8                                                            ______________________________________                                    

The data in Tables 1, 2, 3 and 4 as a whole demonstrate thatparacetamol, contrary to indomethacin, possesses a peculiar selectivityof action on the nervous system, which makes the drug particularlyuseful in the treatment of Alzheimer's disease.

What is claimed is:
 1. A therapeutic method comprising treating apatient suffering from a neurodegenerative disease with atherapeutically effective quantity of a compound having the formula##STR3## wherein R₁ is hydrogen or lower acyl, andR₂ is hydrogen orlower alkyl or a prodrug thereof.
 2. A method according to claim 1,wherein the neurodegenerative disease is Alzheimer's disease.
 3. Amethod according to claim 1, wherein the compound having the formula (I)is paracetamol.
 4. A method according to claim 1, wherein the treatmentcomprises daily administration of 0.01-200 mg/kg of a compound havingthe formula (I) or an equivalent quantity of a prodrug of paracetamol.5. A method according to claim 1, wherein the treatment comprises dailyadministration of 10-100 mg/kg of paracetamol or an equivalent quantityof a prodrug thereof.
 6. A method according to claim 4, wherein thetreatment comprises daily administration of 15-75 mg/kg of paracetamolor an equivalent quantity of a prodrug thereof.